An activation tagging method has been conventionally used as a method for analyzing gene functions. Such method involves producing a mutant with activated transcription of a plant gene with the use of a transcription enhancer sequence incorporated within T-DNA and then cloning the transcriptionally activated gene (Non-patent document 1). A gene that suppresses lateral root formation has been discovered with the use of this method (Patent document 1).
However, the activation tagging method is problematic for use in exhaustive analysis of gene functions (collective analysis of the functions of genes existing in the genome). For example, a 10-Kb genomic region contains an average of 2 or more genes in a model plant such as Arabidopsis. However, in the activation tagging method, an enhancer sequence is used as an activator within a tag and a transcriptionally activatable genomic region has a length of 5 Kb extending beyond each end of an insertion site. Hence, the effects of gene activation with the use of an enhancer are not limited to a single gene, but transcription of a plurality of genes is activated, so that composite phenotypes are generated (Non-patent document 2).
To avoid this problem, the present inventors have developed a technique called the “Fox hunting system (Full length cDNA over-expression gene hunting system)” (Patent document 2). The Fox hunting system is a method that involves introducing a full-length cDNA library as a gene source for strong expression directly in the form of a mixture into a plant through mediation with an Agrobacterium having a strong expression T-DNA vector, seeding the thus obtained T1 seeds, and then screening for phenotypes. When a phenotype of interest appears, a full-length cDNA inserted into the relevant line is examined by PCR and sequencing, thereby resulting in identification of the causative gene.
Examples of the advantages of the Fox hunting system are as described below.
(i) A full-length cDNA library contains all of the amino acid information required for genes to be able to function, so that introduced genes can exhibit their full original functions. Thus, such a full-length cDNA library has much higher efficiency for expressing functions than a general cDNA library. Furthermore, all cDNA fragments are provided with original initiation codon and stop codon information, so that protein fusion is not required for expression and protein expression efficiency is high.(ii) Even if a library of hundreds of millions of clones is used for infection, only 1 to 2 clones are introduced into a plant. Different clones are introduced into transgenic plants, so that fewer instances (roughly two) of confirmation are required for cDNA isolation and phenotypic traits.(iii) With the use of a conventional cDNA library, all the mRNA molecules are replaced by cDNA molecules at their intact quantitative ratios. Hence, the proportions of cDNAs (e.g., structural protein gene groups that are expressed at high levels and signal transduction associated gene groups that are generally expressed at low levels) existing in such cDNA library differ significantly from each other depending on expression level. However, a library that can be used in the Fox hunting system is a “normalized” library that contains all clones in the same proportions regardless of gene expression level. The functions of genes of different species can be tested with efficiency higher than that of genome tagging (However, for plants such as Arabidopsis and rice for which normalized full-length cDNAs have already been established, such cDNAs may also be used. Moreover, when the functions of proteins with high expression levels, such as structural proteins, are analyzed, a general unnormalized full-length cDNA library may also be used).(iv) The functions of all genes within a library can be searched without generating separate lines so as to prepare all plant populations containing full-length cDNAs. Hence, target mutants can be simply obtained and genes (whether or not the functions thereof are the original functions of such genes) imparting specific properties can be screened for with minimal effort.
Early maturation of plants has been reported as follows in Non-patent documents 3 and 4, for example.
At least in the case of Arabidopsis model plants, when a gene referred to as FT is activated within leaves, the mRNA thereof migrates via phloems to shoot apexes, thereby inducing flower buds. Accordingly, flower buds can be induced early via ectopic strong expression of the gene. However, the induction of flower buds requires intracellular gene expression following gene transfer via transformation, so that the preparation of a recombinant is also required herein. Furthermore, it is difficult to control the time during which the relevant gene is expressed; that is, the time during which flower formation is induced.    Patent Document 1: JP Patent Publication (Kokai) No. 2002-010786 A    Patent Document 2: JP Patent Republication (Saikohyo) No. 2003/018808    Non-patent Document 1: Walden, R et al., Plant Mol Biol, 1994, 26(5): pp. 1521-8    Non-patent Document 2: Ichikawa et al., Plant J, 2003, 36: pp. 421-429    Non-patent Document 3: Heang et al., Science 2005, 309, pp. 1694-1696    Non-patent Document 4: Hanzawa et al., PNAS 2005, 102, pp. 7748-7753